X-Message-Number: 17063 From: Date: Sun, 22 Jul 2001 23:58:54 EDT Subject: Suspended Animation of Cells (Including Stem Cells) Cryonet: I was provided with a procedures guide last year by a large cryogenics-based cell storage lab involved with cancer research. The purpose of the guide is quoted as follows: "This information has been compiled to provide a guide for better understanding of the cryogenic preservation process..." I have reason to believe its reproduction for this forum is OK. I think it is worthy of presentation to the Cryonet for several reasons, one of which is to shed light on a recent question asked regarding the reason cell cultures have been more successfully preserved than tissues and organs. While not specifically addressed, some of the reasons can be gleaned from the text of this writing. There are several other reasons that this information may be of interest to some (while it is mostly or completely "old news" to "cryoveterans"). However, I do want to remind those who may be new to this forum that the purpose and function of cryonics, as well as its theory and techniques, are quite distinct from cell culture preservation. For one, this guide regards *suspended animation* which is currently not possible for all but the smallest and "simplest" of multi-cellular animals. While I plan to present most or all of this nine-page manual in sections over the next few days, I will provide a list of all the headings and the complete list of cited Reference at the end of this particular post. Immediately to follow is the Introduction Section only for today's posting. QUOTE: GENERAL GUIDE FOR CRYOGENICALLY STORING ANIMAL CELL CULTURES by John A. Ryan Maintaining healthy, growing cell cultures is a demanding task made more difficult by the ever-present risk of their loss through accidents or contamination. In addition, actively growing cell cultures are not static but, like all populations of microorganisms, subject to age-related or environmentally induced changes which can result in their ongoing evolution and potential loss. These problems are reduced by using cryogenic preservation to stop biological time for cell cultures, effectively putting them into true suspended animation. This concept, long a favorite ploy of science fiction writers and movie producers, has been a reality since the important discovery by Polge, Smith and Parkes (11) in 1949 that glycerol prevents injury to cells caused by freezing. Many cook book-style protocols are now available for freezing cells and these procedures usually perform well (3, 6, 13, 14, 15, 16). It is essential, however, when problems arise or protocol adaptations and improvements must be made, that the underlying concepts on which they are based are well understood. This guide examines both the basic theoretical concepts and practical aspects necessary for successfully freezing animal cells and managing a cell repository. ADVANTAGES OF FREEZING CELL CULTURES Once successfully frozen and stored, cell cultures require little time and effort for their maintenance. The only real cost is the expense of maintaining an ultracold (-130C or lower) mechanical freezer or liquid nitrogen supply. This limited expense compares very favorably with the time, effort and substantial cost of the media and supplies necessary for maintaining actively growing cultures, or for the cost of obtaining a new culture from a repository. Frozen cultures provide an important backup supply for replenishing occasional losses due to contamination or accidents and provide the assurance of a homogeneous culture supply. Cellular changes or alterations occur in all actively growing populations. These changes often result in the loss of important characteristics during evolution of the cultures thereby introducing unwanted variables into long-term experiments. Cryogenically preserved cultures apparently do not undergo any detectable changes once they are stored below -130C (1, 8). Therefore, the biological effects of in vitro cellular aging and evolution may be minimized by frequently returning to frozen stock cultures, allowing ongoing long-term culture experiments to be successfully completed without these unwanted variables. Frozen cultures also provide a valuable baseline against which future experimentally induced changes may be compared or measured. GENERAL EVENTS DURING CELL FREEZING To understand why freezing protocols work, it is necessary to examine both the intracellular and extracellular events occurring in animal cell cultures during the freezing process (2, 4, 8). Initial cooling from room temperature to 0 degrees slows cellular metabolism, rapidly disrupting active transport and ionic pumping. Usually this disruption does not result in cellular damage if the culture medium is osmotically balanced. As cooling continues (0C to -20C) ice crystals begin to form in the extracellular environment which increases the solute concentrations of the culture medium as a result, water begins to move out of the cells and into the partially frozen extracellular medium, beginning the process of cellular dehydration and shrinkage. When the cooling process is rapid, intracellular ice crystals form before complete cellular dehydration has occurred. These ice crystals disrupt cellular organelles and membranes and lead to cell death during the recovery (thawing) process. When the cooling process is slow, free intracellular water is osmotically pulled from the cells resulting in complete cellular dehydration and shrinkage. This can also cause cellular death but there is little agreement on the mechanisms involved. The physical stresses of cellular shrinking may cause some damage resulting in irreparable membrane loss and cytoskeletal and organelle disruption. Damage may also be caused by the high concentrations of solutes in the remaining unfrozen extracellular medium (essentially a brine solution). These solutes attack cells both externally and internally, resulting in membrane damage, pH shifts and general protein denaturation. However, when the cooling rate is slow enough to prevent intracellular ice formation, but fast enough to avoid serious dehydration effects, cells may be able to survive the freezing and thawing process. This survival zone or window is readily observed in many bacterial and other prokaryotes, but for most eukaryotic cells it is nonexistent or very difficult to find without using cryoprotective agents. These agents have little effect on the damage caused by fast freezing (intercellular ice crystal formation), but neither prevent or lessen the damage caused by slow freezing (dehydration and shrinkage) (8). The final storage temperature is also critical for successful cryopreservation. To completely stop biological time, storage temperatures must be maintained below -130C, the glass transition point below which liquid water does not exist and diffusion is insignificant. While many cell cultures are successfully stored at -70C to -90C for months or even years, biological time is not stopped, only slowed, and cellular damage or changes will accumulate. Storage in liquid nitrogen at -196C effectively prevents all thermally driven chemical reactions. Only photo-physical effects caused by background ionizing radiation still operate at this temperature. Thousands of years are estimated to be necessary before background radiation will have a noticeable effect on cryopreserved cultures (2, 8). PRACTICAL ASPECTS OF CELL FREEZING Under the best of circumstances the process of freezing remains stressful to all cell cultures. It is important that everything possible be done to minimize these stresses on the cultures in order to maximize their subsequent recovery and survival. The following suggestions and recommendations are designed to augment of the protocols referred to earlier. ******************************************************** Near future Section Headings to appear over the next few days are: 1. Cell Selection, 2. Cell Harvesting, 3. Cryoprotection, 4. Storage Vessels, 5. Labeling and Recordkeeping, 6. Cooling Rate, 7. Cryogenic Storage, 8. Thawing, 9. Recovery, 10. Problem Solving Suggestions, 11. Managing a Cell Repository ******************************************************** REFERENCES 1. Aswood-Smith, M.J. and G.B. Friedmann, 1979. Lethal and Chromosomal Effects of Freezing, Thawing, Storage Time and X-irradiation on Mammalian Cells Preserved at -196C in Dimethylsulfoxide. Cryobiology 16:132-140 2. Aswood-Smith, M.J., 1980. Low Temperature Preservation of Cells, Tissues and Organs, p. 19-44. In Low Temperature Preservation in Medicine and Biology. M.J. Aswood-Smith and J. Farrant, Eds. (Pitman Medical Limited, Kent, England). 3. Coriell, L.L., 2979. Preservation, Storage and Shipment, p. 29-35. In Methods in Enzymology. Vol. 58, W.B. Jacoby and I.H. Pasten, Eds., (Academic Press, New York). 4. Farrant, J., 1989. General Observations on Cell Preservation, p. 1-18. In Low Temperature Preservation in Medicine and Biology, M.J. Aswood-Smith and J. Farrant, Eds. (Pitman Medical Limited, Kent, England). 5. Freshney, R.L., 1994. Culture of Animal Cells: A manual of Basic Technique, p. 254-263. (3rd edition; Wiley-Liss, New York. 6. Hay, R.J., 1978. Preservation of Cell Culture Stocks in Liquid Nitrogen, p. 787-790. TCA Manual 4. 7. Klebe, R.J. and M. G. Mancuso, 1983. Identification of New Cryoprotective Agents for Cultured Mammalian Cells. In Vitro 19:167-170. 8. Mazur, P., 1984. Freezing of Living Cells; Mechanisms and Implications, p. C125-C142. Am. J. Physiol. 247 (Cell Physiol. 16). 9. McGarrity, G.J., J. Sarama, and V. Vanaman, 1985. Cell Culture Techniques. ASM News 51:170-183. 10. Peterson, W.D., W.F. Simpson and B. Hukku, 1973. Cell Culture Characterization: Monitoring for Cell Identification, p. 164-178. In Tissue Culture: Methods and Applications, P.F. Kruse and M.K. Patterson, Jr. Eds. (Academic Press, New York). 11. Polge, C., A. U. Smith, and A.S. Parkes, 1949. Revival of Spermatozoa after Vitrification and Dehydration at Low Temperatures. Nature 164: 666 12. Ryan, J., 1994. Understanding and Managing Cell Culture Contamination, TC-CI-559. Corning Costar Corporation Technical Monograph. 13. Schroy, C.B., and P. Todd, 1976. A Simple method for Freezing and Thawing Cultured Cells, p. 309-310. TCA Manual 2, Procedure Number 76035. 14. Shannon, J.E. and M.L. Macy, 1973. Freezing, Storage, and Recovery of Cell Stocks, p. 712-718. In Tissue Culture: Methods and Applications. P.F. Kruse and M.K. Patterson, Jr. Eds. (Academic Press, New York). 15. Smith, K.O., 1981. Low Temperature Storage of Surface Attached Living Cell Cultures. Cryobiology 18:251-257. 16. Waymouth, C. and D. S. Varnum, 1976. Simple Freezing Procedure for Storage in Serum-free Media of Cultured and Tumor Cells of Mouse, p. 311-313. TCA Manual 2, Procedure Number 76165. UNQUOTE David C. Johnson, Raleigh, NC Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=17063